Vol. 48, No. 1JOURNAL OF VIROLOGY, OCt. 1983, p. 314-3190022-538X/83/100314-06$02.00/0Copyright © 1983, American Society for Microbiology

Terminal Amino Acid Sequences and Proteolytic CleavageSites of Mouse Mammary Tumor Virus env Gene ProductsLOUIS E. HENDERSON,* RAYMOND SOWDER, GARY SMYTHERS, AND STEPHEN OROSZLAN

LBI-Basic Research Program, Laboratory of Molecular Virology and Carcinogenesis, National CancerInstituite-Frederick Cancer Research Facility, Frederick, Malrland 21701

Received 11 May 1983/Accepted 19 July 1983

The mature envelope glycoproteins of mouse mammary tumor virus (gp52 andgp36) were isolated by reversed-phase high-pressure liquid chromatography. TheN-terminal amino acid sequence of gp36 was determined for 28 residues. The C-terminal amino acid sequences of gp52 and gp36 were determined by carboxypep-tidase digestion. The N-terminal amino acid sequence of gp52 has been reportedpreviously (L. 0. Arthur et al., J. Virol. 41:414-422, 1982). These data werealigned with the predicted amino acid sequence of the env gene product obtainedby translation of the DNA sequence (S. M. S. Redmond and C. Dickson, Eur.Mol. Biol. Org. J. 2:125-131, 1983). The amino acid sequences of the mature viralproteins were in agreement with the predicted amino acid sequence of the envgene product over the regions of alignment. This alignment showed the sites ofproteolytic cleavages of the env, gene product leading to the mature viral envelopeglycoproteins. The N-terminal amino acid sequence of gp52 starts at residue 99 ofthe predicted structure indicating proteolytic cleavage of a signal peptide. Adipeptide (Lys-Arg) is excised between the C-terminus of gp52 and the N-terminus of gp36. The C-terminal amino acid sequence of gp36 is identical to thesequence predicted by the codons immediately preceding the termination codonfor the env gene product. The data show that there is no proteolytic processing atthe C-terminal of the murine mammary tumor virus enm gene product and that theenv gene coding region extends into the long terminal repeat.

Mouse mammary tumor virus (MMTV) is theprototype for the B-type virus of the RNAretroviral family. The synthesis, structure, andbiology of MMTV have recently been reviewed(6). During reverse transcription of viral RNA,regions of the 5' and 3' ends are copied twice,resulting in a long terminal repeat (LTR) on thelinear viral DNA transcript and in the integratedproviral DNA. The gene order of MMTV linearviral DNA transcript from 5' to 3' ends is LTR-gag-pol-env-LTR where gag, poi, and env aregenes coding for known viral proteins. The gaggene codes for a precursor polyprotein (Pr77g"9)which is post-translationally modified by phos-phorylation (19) and myristylation (26). The polgene codes for reverse transcriptase. The enmgene codes for a precursor polyprotein (Pr69'"')which is cotranslationally transported into theendoplasmic reticulum where a signal peptide isremoved by proteolysis (4) and post-translation-al glycosylation is initiated. The glycosylatedprecursor (gPr73e"') is transported to the cellmembrane where it participates in viral assem-bly and is subsequently cleaved by proteolysisto viral gp52 and gp36 (4, 25). These two glyco-proteins are associated with the lipid envelope of

the mature virus. Gp36 is believed to be atransmembrane glycoprotein. Gp52 probably de-termines the tissue tropism of the virus byinteracting with specific cell surface receptorsduring viral infection. In addition, MMTV glyco-proteins show immunological cross-reactivitywith antigens associated with breast cancer (3,18, 20).Co- and post-translational modification of the

viral precursor polyprotein may be importantcontrolling events in the viral assembly andmaturation process. The nature of some of thesemodifications can be directly determined bystructural studies on the viral proteins. Thenature of post-translational proteolytic cleav-ages can best be determined by comparing ami-no acid sequences of the mature viral protein tothe nucleic acid sequence of the viral genome.The nucleotide sequence of MMTV (C3H)

LTR (9, 16) and of two fragments of MMTV(GR) proviral genome including the entire enmgene (23) and part of the LTR (10, 23) has beendetermined. In the LTR region where the knownnucleotide sequences of the two strains can becompared, there are only 60 base changes scat-tered over 1,360 nucleotides suggesting minimal

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NOTES 315

E

CDCM

00i

.5

cn

c._

I co

0

0,-

0

C- =

h.C020a,

mlFIG. 1. Separation ofMMTV (C3H) structural proteins by RP-HPLC. A 2.0-mg amount ofMMTV (C3H) was

suspended in 10 ml of water and adjusted to pH 2.0 with trifloroacetic acid and added to 20 ml of saturatedguanidine-HCl. The mixture was shaken for 1 min and then applied to a column (0.39 by 30 cm) of p.BondapakC18 at a flow rate of 1.0 ml/min. After sample application, the column was washed with 40 ml of 0.1%trifloroacetic acid at 1.0 ml/min (data not shown). Elution was accomplished at 1.0 ml/min and 23°C withgradients of increasing acetonitrile (0.1% trifloroacetic acid) (---) as follows: 0 to 20%, 10 min; 20 to 50%, 120 min;50 to 60%, 10 min. The column temperature was changed to 52°C and the organic solvent changed to 1-propanol(0.1% trifloroacetic acid), and elution continued with increasing gradients as follows: 30% isocratic, 10 min; 30 to100o, 60 min. Eluted components were detected by UV absorption at 206 nm. Peaks 1 through 10 were analyzedby SDS-PAGE as shown in Fig. 2.

structural differences between the two strains.The nucleotide sequence shows three possibleinitiation codons in the same open reading framecoding for the env gene product (23). It is notknown which of these is the initiation codon forthe synthesis of p69env in vitro. Therefore, theDNA sequence was translated into amino acidsequence starting at the first possible initiationcodon and the predicted amino acid sequence ofp69env, numbered accordingly (23). Structuralstudies on the viral env gene products (gp52 andgp36) from either strain should elucidate theexact coding region for these proteins and defineproteolytic processing events that lead to thefinal viral products. Here, we report the N-terminal amino acid sequence of gp36 (C3H) forthe first 28 residues and the C-terminal aminoacid sequences of gp52 (GR) and gp36 (GR). TheN-terminal amino acid sequence of gp52 (C3H)was also determined in this laboratory and re-ported previously (1). These data are comparedwith the amino acid sequence of the MMTV envprecursor predicted from the nucleotide se-quence of the env gene (23) and the LTR (9, 16).The comparison reveals the proteolytic cleavagesites in the precursor between gp52 and gp36 andshows that the N-terminal amino acid sequenceof gp36 (C3H) is identical to the sequence pre-dicted from the nucleotide sequence of the GRstrain. The comparison also shows that the C-terminal region of the viral gp36 is coded bynucleotide sequences which extends 83 basesinto the LTR. This is the first observation that

nucleotides in retroviral LTR structures cancode for protein synthesis.MMTV structural proteins were purified by

reversed-phase high-pressure liquid chromatog-raphy (RP-HPLC) by using techniques similar tothose previously described for the purification oftype C viral proteins (13, 14). Fig. 1 shows theseparation of the MMTV (C3H) structural pro-teins by RP-HPLC. The viral proteins wereseparated by gradient elution at pH 2 as indicat-ed in Fig. 1. Column fractions 1 through 6 wereeluted with acetonitrile gradients at 23°C. Frac-tions 7 through 10 were eluted with a 1-propanolgradient at 50°C as indicated in Fig. 1. Portionsof each fraction were analyzed by sodium dode-cyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) (Fig. 2). Column fraction 1 did notcontain any protein detectable by Coomassie-stained SDS-PAGE analysis. Fraction 2 con-tained highly purified gp52 (Fig. 2, lane 2),fraction 4 contained purified p27, fraction 5contained purified plO, and fraction 6 containedadditional p27. Fractions 7 and 10 did not con-tain any detectable protein. Fraction 8 containedlipids that migrate near the dye front on SDS-PAGE and are detectable with Coomassie stain-ing. Fraction 9 contained gp36 and lipids (Fig. 2,lane 9). The separation of the structural proteinsof MMTV (GR) was identical to the separationshown in Fig. 1. In the preparations of MMTVused, both p14 and p21 were present in minoramounts compared with the amount of gp52,p27, plO, and gp36. In other separations by the

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same techniques but with larger inputs of virus(ca. 10 mg), p14 and p21 were detected (data notshown). P14 eluted in about 25% acetonitrile andp21 eluted in about 31% acetonitrile.Amino acid analysis of purified gp36 (GR) is

given in Table 1. The data are given as moles ofeach amino acid per mole of protein, calculatedon the assumption that the peptide chain con-tains 232 amino acid residues as predicted by thenucleotide sequence of the env gene product(GR) and the defined coding region for gp36 asdeduced from the data given in this report. Thepredicted amino acid composition of gp36 (GR)is also given in Table 1. The experimentallydetermined values for each residue are within10% of the predicted values for all residuesexcept methionine and isoleucine. The low val-ues obtained for methionine and isoleucine areprobably due to low recoveries of the residuesafter acid hydrolysis.Table 2 gives the results of semiautomated

Edman degradation of gp36 (C3H). The N-termi-nal amino acid sequence was determined for 28residues (Table 2). No phenylthiohydantoin(PTH) amino acid was detected in cycle 18. Allother assignments listed in Table 2 were unam-biguous.The C-terminal amino acid sequence of gp36

(GR) was determinedl by carboxygestion by methods previously deQuantitative data for the amino aby digestion with either carboxyp4

S I

FIG. 2. Shows the results of SDS-]of RP-HPLC fractions 1 through 10 (110) from Fig. 1. Molecular weight stanLaboratories) were run in lane S. Thperformed by the method of Laemmligradient gel (15). Proteins were visualizsie staining.

TABLE 1. Amino acid analysis of gp36 (GR)

Residues per mole

Amino acid Predicted fromnucleotide se- Found"

quence

Asp + Asn 26 27.1Thr 14 15.4Ser 12 13.5Glu + Gln 23 23.0Pro 6 6.6Gly 9 9.5Ala 25 25.3Val 19 17.6Met 5 3.8Ile 19 15.1Leu 33 32.1Tyr 5 5.5Phe 9 8.4His 4 4.1Lys 10 9.9Arg 8 8.1Trp 3 NDCys 3 ND

a Assuming a molecular weight of 26,000 for thepeptide chain. Results based on 24-h hydrolysis of theprotein and a recovery of 85% for Thr and 80% for Ser.ND, Not determined.

'peptilase 1- carboxypeptidase B are given in Table 3. These,scribed (22). results show that the C-terminal amino acidcids released sequence of gp36 (GR) is -Arg-Val-Ser-Tyr-Thr-eptidase A or OH.

The data given in Tables 2 and 3 were used to9 lw establish the gp36 coding region in the predicted

amino acid sequence of the env gene product(23). The experimentally determined N-terminalamino acid sequence of mature viral gp36 (C3H)given in Table 2 is identical to the amino acidsequence predicted from nucleotide sequencesfor both C3H strain (H. E. Varmus, personalcommunication) as well as the GR strain (23).

4i Residue 18 was not identified by Edman degra-* - gp: dation of the mature viral protein and is a Ser in

the predicted sequence. The low recovery of-&-' PTH-Ser or a strain difference may account for

the failure to detect a PTH amino acid in Edmancycle 18, but the possibility of a post-translation-ally modified Ser at this position cannot beexcluded. The experimentally determined C-terminal amino acid sequence of mature viralgp36 (GR) is identical to the sequence fromresidue 684 to the C-terminal end (residue 688)of the predicted sequence of the env gene prod-

PAGE analsIs.uct of MMTV GR (23) and also of MMTV C3Hanes 1 thraoyusgh (H. E. Varmus, personal communication). Thedards (Bio-Rad amino acid composition of viral gp36 (GR) is ine analysis was good agreement with the amino acid composi-on a 10 to 20% tion of residues 457 through 688 in the predicteded by Coomas- sequence of the env gene product (GR) (Table

1). The alignment of the viral gp36 amino acid

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2 1. 15) k - ...,..4

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TABLE 2. Amino terminal amino acid sequence ofMMTV gp36 (C3H)'

Cycle Nanomoles Cycle NanomolesCycle ~of PTH Cce of PITHno. amino acid no. amino acid

1 Phe (4.25) 15 Ile (0.90)2 Val (3.71) 16 Ile (0.92)3 Ala (3.09) 17 Thr (0.60)4 Ala (2.46) 18 X5 Ile (2.45) 19 Phe (0.78)6 Ile (2.64) 20 Ala (0.70)7 Leu (1.98) 21 Val (1.20)8 Gly (1.21) 22 Ala (1.03)9 Ile (2.02) 23 Thr (0.39)10 Ser (0.37) 24 Thr (0.39)11 Ala (1.25) 25 Ala (0.93)12 Leu (0.85) 26 Leu13 Ile (1.50) 27 Val14 Ala (1.19) 28 Lys

a Sequence was determined by semiautomatic Ed-man degradation of 4.5 nm of MMTV gp36 (C3H).PTH amino acid was identified by RP-HPLC as previ-ously described (11). X indicates no PTH amino acididentified in cycle 18 (see text).

sequence data with the nucleotide sequence datais shown schematically in Fig. 3. This alignmentreveals that 83 nucleotides at the 5' end of theLTR region are within the coding region for thegp36 protein. The nucleotide sequences ofMMTV (GR) (10, 23) and MMTV (C3H) LTR (9,16) segments coding for the C-terminal region ofgp36 are identical with the exception of a singlebase change which does not change the transla-tion of the codon. A long open reading frame inthe nucleotide sequence of MMTV LTR hasbeen noted, and it has been suggested thatsequences in the LTR may direct protein synthe-sis (9, 10, 16). The initiation codon for this longopen reading frame in the LTR is located 53nucleotides upstream from the termination co-don at the end of the env gene. Thus, the longopen reading frame in the LTR overlaps the envgene but is not in the same reading frame as theenv gene. Therefore, the env gene productsdiscussed here are not directly related to thesuggested translational products of the LTR longopen reading frame. However, the fact that theC-terminal portion of gp36 is coded by nucleo-tides in the LTR shows that nucleotides in theLTR can direct protein synthesis.The first 47 amino acid residues in the amino

acid sequence of MMTV gp52 (C3H) have beendetermined by direct analysis of the protein andhave been previously reported (1). This se-quence is identical to residues 99 through 145 inthe predicted sequence of the env gene product(GR) with the exception of two residues. Thenucleotide sequence predicts a Ser at position118 and a Met at position 130 in the env gene

product of the GR strain, and the amino acidsequence studies identified Asn and Ile, respec-tively, at these positions in the C3H strain.Digestion of gp52 (GR) for 1 h with carboxypep-tidase B released 0.34 mol of alanine, 0.31 mol ofarginine, and 0.26 mol of isoleucine per mol ofprotein. These results are consistent with a C-terminal sequence Ile-Arg-Ala-OH for viralgp52. This sequence is found at residues 452through 454 in the predicted amino acid se-quence for the env gene product (GR) (23).These data, when compared with the nucleotidesequence of the env portion of the proviral DNA(23), define the initial and final coding regionsfor the fully processed gp52 (Fig. 3).

Fig. 3 depicts the known nucleotide sequenceof the 3' end of the MMTV genome starting atthe first initiation codon in the env gene andsummarizes the co- and post-translational pro-teolytic cleavages of the env gene product thatare shown here to occur during the synthesis andprocessing of viral envelope proteins.The biosynthetic process for p69env (7) and

gPr73env (5) has been recently reviewed (6).Proteolytic cleavage of a 9,000-dalton signalpeptide from the N-terminal end of p69env isthought to be a cotranslational event accompa-nying insertion of the nascent peptide into theendoplasmic reticulum where glycosylation isinitiated. The N-terminal amino acid residues ofp69env and gPr73env have not been determined.Therefore, the exact initiation site for p69envsynthesis and the nature and number of proteo-lytic events leading ultimately to the N-terminalend of the fully processed gp52 are not known.The scheme represented in Fig. 3 assumes acommon N-terminal residue *for gp73en" andgp52 (residue 99 in the predicted sequence ofp699env). The C-terminal amino acid sequence ofgp36 is identical to the predicted C-terminalsequence for p69enV; therefore, there is no pro-

teolytic processing at the C-terminal end of theenv gene products of MMTV. This is in contrastto type C murine viruses in which proteolyticprocessing at the C-terminal end of the env geneproducts is known to occur (21, 27). Proteolyticprocessing of gPr73env to gp52 and gp36 involves

TABLE 3. Residues released by carboxypeptidasedigestion of MMTV gp36 (GR)

Picomoles of amino acid released'Digestion

Arg Val Ser Tyr Thr

Carboxypeptidase A 25.7 31.6 90.7 92.4Carboxypeptidase B 9.2 14.1 32.8 91.3 93.5

a In each experiment 132 pmol of gp36 (GR) digest-ed as previously described (22)., The protein concen-tration was determined by amino acid analysis as inTable 1.

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TAG

688-l p69env predicted sequence

'

_1"1

I

/1/1

199 4541 157 688

-Thr-Gly-Gl u-Ser-ITy /v Il e-Arg-Al a-Lys-Arg-Phe-Val -Al /V// Ser-Tyr-Thr-Stop

NH2-G1 u-SerTyr-'Tyr. Il e-Arg-Al a-OH NH2-Phe-Val -Al a-/\//\/\/\Ser-Tyr-Thr-OH

iViral gp52 1 Vi-ral gp36

FIG. 3. A schematic representation of the synthesis and proteolytic processing of the MMTV ens' gene

product. The nucleotide sequences of the env (23) and LTR (9) regions of the genome are represented by the firstline in the diagram. An open reading frame extends from the first ATG initiation site through two more ATGcodons and ends at a TAG termination codon which is located 84 nucleotides into the LTR as shown. The secondline of the diagram represents the predicted amino acid sequence of the ens' gene product (p69e"' ), assuming thatprotein synthesis initiates at the first ATG codon. Numbers indicate the position of amino acid residue in thepredicted sequence of p69e"'. Arrows indicate proven proteolytic cleavage sites. The predicted amino acidsequences around the cleavage sites and at the C-terminal end of p69'"' are shown and compared to the N- and C-terminal amino acid sequences of the mature gp52 and gp36. Intervening sequences not shown are indicated bysaw-toothed lines.

the removal of a Lys-Arg sequence as indicatedin Fig. 3. An identical sequence is found in theenv precursor of type C murine virus (27) whereproteolytic cleavage at the C-terminal side ofArg is known to take place (21). Thus, thecleavage sites between gp52 and gp36 of MMTVbear a strong resemblance to their functionalcounterpart in murine type C viruses. In the caseof Friend murine leukemia virus, the Lys-Argsequence is retained at the C-terminal end of theglycoprotein (2), whereas in the Rauscher andMoloney strains, most of the isolated glycopro-tein has these residues removed, possibly by theaction of carboxypeptidases (L. E. Henderson,T. D. Copeland, G. W. Smythers, R. Sowder,and S. Oroszlan, unpublished data).Gp36 is known to be a hydrophobic mem-

brane-associated protein (8, 17). Its hydrophobicnature is indicated by its strong binding to thereversed-phase support (Fig. 1) which facilitatesits isolation by RP-HPLC. Similar strong bind-ing to reversed-phase supports is observed fortype C membrane-associated homologs of gp36(i.e., pl5E of murine leukemia viruses) (13, 14).Ongoing work on the type B and type C trans-membrane proteins indicates that additionalpost-translational modification may result in thecovalent attachment of strongly hydrophobicmoieties to these proteins. The exact chemicalnature of these hydrophobic moieties is un-known, but it is probable that they involve fattyacid acylation at serine or threonine hydroxylgroups such as has been proposed for the enve-

lope proteins of vesicular stomatitis and Sindbisvirus (24). This type of fatty acid acylation isdifferent from the well-documented covalent at-tachment of a long-chain fatty acid (myristicacid) to the N-terminal residue of the type C (12)and type B gag precursors (26). Continued workon the nature and function of these hydrophobicpost-translational modifications of the gag andenv proteins may reveal important steps in themechanisms of virus assembly and budding.Moreover, these post-translational events mayhave some relationship to the oncogenic poten-tial of these viruses.

This research was sponsored by the National Cancer Insti-tute, Department of Health and Human Services, undercontract N01-CO-23909 with Litton Bionetics, Inc.

LITERATURE CITED

1. Arthur, L. O., T. D. Copeland, S. Oroszlan, and G.Schochetman. 1982. Processing and amino acid sequenceanalysis of the mouse mammary tumor virus env geneproduct. J. Virol. 41:414-422.

2. Chen, R. 1982. Complete amino acid sequence and glyco-sylation of glycoprotein gp71A of Friend murine leukemiavirus. Proc. Natl. Acad. Sci. U.S.A. 79:5788-5792.

3. Day, N. K., S. S. Witkin, N. H. Sarkar, D. Kinne, D. J.Jussawalla, A. Levin, C. C. Hsia, N. Geller, and R. A.Good. 1981. Antibodies reactive with murine mammarytumor virus in sera of patients with breast cancer: Geo-graphic and family studies. Proc. Natl. Acad. Sci. U.S.A.78:2483-2487.

4. Dickson, C., and M. Atterwill. 1979. Composition, ar-rangement and cleavage of the mouse mammary tumorvirus polyprotein precursor Pr77eae and p110"'5. Cell17:1003-1012.

5. Dickson, C., and M. Atterwill. 1980. Structure and proc-

ATGCodons prov i ra 1

DNA

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essing of the mouse mammary tumor virus glycoproteinprecursor Pr73e"'. J. Virol. 35:349-361.

6. Dickson, C., R. Eisenman, H. Fan, E. Hunter, and N.Teich. 1982. Protein biosynthesis and assembly, p. 513-648. In R. A. Weiss, N. M. Teich, H. E. Varmus, and J.M. Coffin (ed.), Molecular biology of tumor viruses, 2nded., RNA tumor viruses. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

7. Dickson, C., and G. Peters. 1981. Protein coding potentialof mouse mammary tumor virus genome RNA as exam-ined by in vitro translation. J. Virol. 37:36-47.

8. Dion, A. S., C. J. Williams, and A. A. Pomenti. 1977. Themajor structural proteins of murine mammary tumor vi-rus: techniques of isolation. Anal. Biochem. 82:18-28.

9. Donehower, L. A., A. L. Huang, and G. L. Hager. 1981.Regulatory and coding potential of the mouse mammarytumor virus long terminal redundancy. J. Virol. 37:226-238.

10. Fasel, N., K. Pearson, E. Buetti, and H. Diggelmann. 1982.The region of mouse mammary tumor virus DNA contain-ing the long terminal repeat includes a long coding se-quence and signals for hormonally regulated transcrip-tion. Eur. Mol. Biol. Org. J. 1:3-7.

11. Henderson, L. E., T. D. Copeland, and S. Oroszlan. 1980.Separation of all amino acid phenylthiohydantoins byhigh-performance liquid chromatography on phenylalkylsupport. Anal. Biochem. 102:1-7.

12. Henderson, L. E., H. C. Krutzch, and S. Oroszlan. 1983.Myristyl amino terminal acylation of murine retroviralproteins: an unusual post-translational protein modifica-tion. Proc. Natl. Acad. Sci. U.S.A. 80:339-343.

13. Henderson, L. E., R. Sowder, and S. Oroszlan. 1981.Protein and peptide purification by reversed-phase high-pressure chromatography using volatile solvents. p. 251-260. In D. T. Liu, A. N. Schechter, R. Heinriksson, andP. G. Condliffe (ed.), Chemical synthesis and sequencingof peptides and proteins. Elsevier, Amsterdam.

14. Henderson, L. E., R. Sowder, and S. Oroszlan. 1982.Reversed phase (RP) HPLC of proteins and peptides andits impact on protein microsequencing, p. 409-416. In M.Elzinga (ed.), Proceedings of the IV international confer-ence on methods in protein sequence analysis. The Hu-mana Press, Inc., Clifton, New Jersey.

15. Laemmli, U. K. 1970. Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4.Nature (London) 227:680-685.

16. Majors, J., and L. Donehower. 1982. Appendix D: se-quences of retroviral LTRs, p. 1333-1336. In R. A. Weiss,N. M. Teich, H. E. Varmus, and J. M. Coffin (ed.),

Molecular biology of tumor viruses, 2nd Ed., RNA tumorviruses. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

17. Marcus, S. L., S. W. Smith, J. Racevskis, and N. H.Sarkar. 1978. The relative hydrophobicity of oncornaviralstructural proteins. Virology 86:398-412.

18. Masa-Tejada, R., I. Keydar, M. Ramanarayanan, T.Ohno, C. Fenoglio, and S. Spiegelman. 1979. Detection inhuman breast carcinomas of an antigen immunologicallyrelated to a group-specific antigen of mouse mammarytumor virus. Proc. Natl. Acad. Sci. U.S.A. 75:1529-1533.

19. Nusse, R., F. A. M. Asselbers, M. H. Salden, R. J. A. M.Michalides, and H. Bloemendal. 1978. Translation ofmouse mammary tumor virus RNA: precursor polypep-tides are phosphorylated during processing. Virology91:106-115.

20. Ohno, T., R. Masa-Tejada, I. Keydar, M. Ramanar-ayanan, J. Bausch, and S. Spiegelman. 1979. Humanbreast carcinoma antigen is immunologically related to thepolypeptide of the group-specific glycoprotein of mousemammary tumor virus. Proc. Nat]. Acad. Sci. U.S.A.76:2460-2464.

21. Oroszlan, S., L. E. Henderson, T. D. Copeland, A. M.Schultz, and E. Rabin. 1980. Processing and structure ofmurine leukemia virus gag and ent' gene encoded polypo-teins, p. 219-233. In G. Koch, and D. Richter (ed.),Biosynthesis, modification and processing of cellular andviral polyproteins. Academic Press., Inc., N.Y.

22. Oroszlan, S., L. E. Henderson, J. R. Stephenson, T. D.Copeland, C. W. Long, J. N. Ihie, and R. V. Gilden. 1978.Amino- and carboxyl-terminal amino acid sequences ofproteins coded by gag gene of murine leukemia virus.Proc. Natl. Acad. Sci. U.S.A. 75:1404-1408.

23. Redmond, S. M. S., and C. Dickson. 1983. Sequence andexpression of the mouse mammary tumor virus enii gene.Eur. Mol. Biol. Org. J. 2:125-131.

24. Schlesinger, M. J., and C. Malfer. 1982. Cerulenin blocksfatty acid acylation of glycoproteins and inhibits vesicularstomatitis and sindbis virus particle formation. J. Biol.Chem. 257:9887-9890.

25. Schochetman, G., S. Oroszlan, L. Arthur, and D. L. Fine.1977. Gene order of the mouse mammary tumor virusglycoproteins. Virology 83:72-83.

26. Schultz, A. M., and S. Oroszlan. 1983. In vivo modifica-tion of retroviral gag gene encoded polyproteins by myris-tic acid. J. Virol. 46:355-361.

27. Shinnick, T. M., R. A. Lerner, and J. G. Sutcliffe. 1981.Nucleotide sequence of Moloney murine leukemia virus.Nature (London) 293:543-548.

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